Nucleic acid amplification and detection typically requires extensive sample preparation and nucleic acid extraction procedures utilizing laboratory equipment, followed by amplification of the extracted nucleic acid and detection of the amplification product which requires additional equipment.
Detection of nucleic acid without extensive sample preparation simplifies the process and shortens the time from sample-to-answer. This may allow more rapid detection of naked nucleic acid, genetic markers or pathogenic microorganisms in clinical, food testing, agricultural, environmental and field samples. For example, Loop-mediated isothermal amplification (LAMP) is one example technique that does not require extensive sample prep or nucleic acid isolation. LAMP was first described in the article “Loop-mediated Isothermal Amplification of DNA” by Notomi, et al., 2000, Nuc Ac Res, 28(12):e63, and is an isothermal technique which amplifies a target sequence at a constant temperature using either two or three sets of primers, and a DNA polymerase with high strand displacement activity in addition to replication activity (e.g. DNA polymerase from Bacillus stearothermophilus (Bst pol), which has optimal activity at 60-65° C.). Typically LAMP utilizes four different primers: forward and reverse outer primers, F3 and B3 respectively, and forward and reverse inner primers, FIP and BIP, respectively, that target six distinct sequences on the template nucleic acid. The addition of reverse transcriptase into the reaction, termed reverse-transcription LAMP or RT-LAMP, allows for the detection of RNA templates under the same conditions. Additionally, the addition of loop primers was subsequently shown to increase the rate of the reaction, reducing overall amplification times significantly (Nagamine, et al., 2002, Mol Cell Probes, 16:223-229). Thus a complete set of LAMP primers includes: outer primers F3 and B3, inner primers FIP and DIP, and forward and reverse loop primers, LF and LB, respectively.
Various detection methods have been reported for LAMP, including turbidity, fluorescence and gel electrophoresis (reviewed in Panda, 2008, Rev Med Virol, DOI: 10.1002/rmv.593). Additionally, colorimetric detection of positive LAMP reactions using Hydroxynaphthol blue dye (HNB) was described in an article by Goto, et al., 2009, Biotechn, 46(3):167-172. Solutions of HNB undergo a color change as cation levels drop (Brittain 1978, Analyt Chim Acta, 96:165-170). LAMP reactions generate a significant amount of pyrophosphate byproduct as supplied 2′-Deoxyribonucleotide-5′-Triphosphates (dNTPs) are added to amplification product. The pyrophosphate bonds with free Mg2+ in the reaction mixture, reducing the cation level. This results in the solution undergoing a purple to blue color change easily detectable with the human eye.
Recently, several groups have published LAMP assays for the detection of B. anthracis (Qiao, et al., 2007, Biotechnol Lett, 29:1939-1946; Kurosaki 2009; Hatano, et al., 2010, Jpn J Infect Dis, 63:36-40; Jain, et al., 2011, World J Microbiol Biotechnol, 27:1407-1413). Qiao and coworkers originally reported detection of three gene targets representing the B. anthracis plasmids, pXO1 (pag) and pXO2 (capB), along with a chromosome target (Ba813) using LAMP. They reported a lower limit of detection of 10 spores (Qiao 2007) using fluorescence and gel electrophoresis. Kurosaki, et al. reported detection of three B. anthracis target genes (pag, capB, and sap), again representing the two plasmids and chromosome, respectively. They reported a limit of detection for pag of 10 fg per reaction in ˜30 min using purified DNA and real-time turbidity detection (Kurosaki 2009). Additionally, they reported detecting target DNA from spores isolated from blood of intra-nasally infected mice (Kurosaki 2009). Hatano and coworkers reported detecting 1000 copies of pag and capB target DNA using LAMP along with a low-cost pocket warmer as a heating source (Hatano 2010). Most recently, DNA isolated from spores spiked into soil and talcum powder was detected by LAMP targeting the pag gene on pXO1 (Jain 2011).
Previous reports describing the detection of B. anthracis using LAMP have all used isolated DNA as template, whether extracted using phenol/chloroform (Hatano 2010), commercial kits (Kurosaki 2009) or boiling of spores (Qiao 2007, Kurosaki 2009, Jain 2011). These procedures produce quality DNA preparations suitable for PCR and LAMP, but require a minimum of 1 hour to perform and laboratory equipment such as tabletop centrifuges capable of speeds >10K RPM. Researchers recently showed direct detection of nucleic acid from solid and liquid cultures of B. anthracis without time consuming nucleic acid extraction and purification (Dugan et al. 20012, J Microbiol Methods, 90:280-284). Cultures were either loaded directly into the reaction mixture or diluted in buffer and then loaded into the reaction.